Sj

Fig. 2. The shape of the EPR spectra from 57Fe-(or 56Fe)-DNIC neocuproine (A,B). Doublet spectra above spectra A is low-field part of the latter recorded at higher amplification. The spectra were recorded at 77 K. (From Ref. [25].)

as {3d}7 and the iron center acquires the character of Fe+. In the absence of NO, the Fe2+ ions are not chelated by neocuproine because of rapid formation of water insoluble iron hydroxide complexes [25].

Bathocuproine disulfonate (BCS) is a highly soluble cuproine derivative. Vasodilatory studies of Cys-NO have shown that exogenous copper and iron inhibit the relaxation of precontracted endothelium-denuded rat aorta rings by Cys-NO, and that the inhibitory action of both metal ions is cancelled by BCS [17,18]. The addition of this chelator enhanced the duration of the vessel relaxation induced by Cys-NO in the presence of ascorbate, copper or iron (Fig. 3, curves f,g) [17].

The stability of Cys-NO was also studied in vitro in solutions containing ascorbate, copper or iron. In vitro, BCS protected Cys-NO effectively against catalytic decomposition by both metals but strong iron chelator bathophenanthroline disulfonate (BPDS) protected against ferrous iron only (Fig. 4) [17]. The observations were attributed to full saturation of the coordination sphere in the Fe2+-BPDS complexes (i.e. the binding of three BPDS ligands to each iron) and preventing the iron from participating in redox reactions with Cys-NO [17,18]. In contrast, the monovalent copper ions bind only two BPDS ligands, and leaving the copper atom accessible to small molecules in the solution [26]. The experimental observations suggest that Cys-NO can penetrate into Cu(BPDS)2 complexes and be decomposed via reduction by Cu+ (Fig. 4) [17].

The catalytic decomposition of Cys-NO by copper-BPDS in the presence of BPDS and ascorbate was noticeable down to very low copper concentrations of about 2 ^M (Fig. 4) [17,18]. This makes it questionable to attribute the protection of Cys-NO by cuproine derivatives to their sequestration of intrinsic Cu+. For example, Fig. 5 (curve b) [17] shows that BPDS gives significant protection of Cys-NO in vasodilation experiments in

Krebs buffers. It means that intrinsic copper did not form the main pathway for the decomposition of Cys-NO in these experiments. We estimate the intrinsic copper content to be lower than 2 ^M. Instead, the protection of Cys-NO was attributed to the sequestration and inactivation of intrinsic iron in the solutions. Phrased otherwise, intrinsic ferrous iron rather than intrinsic copper was seen to dominate the decomposition of Cys-NO.

In experiments with nitrosothiols, the presence of spurious quantities of reduced iron and copper is often found to be significant. Sheu et al. [27] investigated the iron and copper levels of sample solutions containing cysteine, glutathione (1-2 mM) or phosphate buffer (100 mM) and reported values of [Fe] ~ 1.8 ^M and [Cu] ~ 0.05 ^M. Therefore, spurious

Fig. 4. Influence of subsequent additions (shown by arrows) of ascorbate, copper and iron on the stability of Cys-NO in the presence of 1 mM bathophenanthroline disulfonate (top) or bathocuproine sulfonate (bottom). Results are expressed as mean ± SE of three experiments [17].

Fig. 4. Influence of subsequent additions (shown by arrows) of ascorbate, copper and iron on the stability of Cys-NO in the presence of 1 mM bathophenanthroline disulfonate (top) or bathocuproine sulfonate (bottom). Results are expressed as mean ± SE of three experiments [17].

iron comfortably exceeded copper (Fig. 4). Under these conditions, only the addition of BPDS would bind the intrinsic iron, extend the lifetime of Cys-NO and thereby make the vessel relaxation last longer.

The addition of BCS also prolongs the vasodilatory action of Cys-NO significantly (Fig. 5, curve c) [17], but the mechanism is very different. As remarked before, the combination of iron, Cys-NO and BCS leads to the formation of BCS-DNIC. This BCS-DNIC is quite stable and long-lived and was shown to induce a long-lasting vasodilation in aortic rings from which the endothelium had been removed. The vasorelaxation observed with BCS-DNIC was sustained significantly longer than that induced by DNIC with phosphate ligands (Fig. 6, curves a,b) [17].